Analyte measurement

ABSTRACT

A method for determining the concentration of an analyte in a sample is disclosed which comprises contacting the sample with a micro electrode which comprises an enzyme capable of reacting with said analyte and a redox mediator which is capable of being converted by being oxidised or reduced by said enzyme once the latter has reacted with the analyte, allowing the analyte to react with the enzyme, then applying a potential across the electrode and measuring the resulting concentration of the converted mediator electrochemically.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of International Patent Application No.PCT/GB03/02150 filed on May 16, 2003.

Statement Regarding Federally Sponsored Research Or Development

Not Applicable

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for determining the concentration ofan analyte in a sample and, in particular, to a method for determiningthe concentration electrochemically.

2. Description of the Related Art

It is particularly valuable to determine the concentration of a specificcomponent in a sample which is of biological origin, for example blood.For this purpose a biosensor is used; use is made of a change in theoxidation state of a mediator which interacts with an enzyme which hasreacted with the analyte to be determined. The oxidation state of themediator is chosen so that it is solely in the state which will interactwith the enzyme on addition of the substrate. The analyte reacts with astoichiometric concentration of the mediator via enzyme. This causes themediator to be oxidised or reduced (depending on the enzymatic reaction)and this change in the level of mediator can be measured by determiningthe current generated at a given potential.

Normally, a measurement is taken during the oxidation (or reduction) ofthe mediator by the enzyme as it reacts with the analyte. This can,though, give rise to unreliable results. It has been proposed,therefore, to wait for the reaction to go to completion and then to takea measurement. However, the value obtained changes with time such thatit is generally necessary to take a number of readings and then todetermine the concentration using either an algorithm or by integrationof the area under the curve which corresponds to the plot of the values.This change in value is caused by the effect of diffusion which occursessentially linearly from the electrode. Thus as some of the mediator isoxidised (or reduced) on the electrode more mediator diffuses to theelectrode on a continuing basis. However, this linear diffusion resultsin depletion of electroactive material around the working electrode.

In order to determine the concentration it is necessary to first of allobtain a value in the absence of the analyte and to subtract this“background” value from the enhanced value which one obtains when theanalyte is present. It will be appreciated that this procedure is bothcomplicated and prone to error.

Recently, steps have been taken to reduce the size of biosensors bymaking use of a micro electrode. This can be defined as an electrodewhere at least one of the dimensions does not exceed 50 μm and isfrequently 1 to 25 or 30 μm.

Micro electrodes were devised because they were perceived to be betterfor the measurement of very small currents. This is because the use ofan array of micro electrodes gives a better signal to noise ratio thandoes a single electrode. Micro electrodes were therefore devised fordirect current determination and they have not found utility asbiosensors involving an enzyme and a mediator. It has, though,surprisingly been found that the use of a micro electrode can, if usedin a particular way, overcome the disadvantages of macro electrodes.

That micro electrodes can be used for this purpose is particularlysurprising for two principal reasons. First, the current enhancement dueto the presence of the analyte over the background level for a microelectrode is of course very much smaller than for a macro electrode.Accordingly with decreasing size it is impossible to obtain any accuratemeasurements. Second, because the area of the electrode is very small inrelation to the volume of the sample diffusion to the electrode surfaceno longer takes place linearly as with macro electrode but, rather,radially. The distance over which the reaction occurs in solution islarge compared to the size of the microelectrodes such that one wouldexpect there to be little or no catalytic enhancement. One wouldtherefore expect with decreasing electrode size no meaningfulmeasurement to be obtainable.

Under normal steady-state conditions, the average distance that theoxidised state of the mediator will diffuse before it reacts to reformthe reduced state will be (Dt_(L))^(1/2) where D is the diffusioncoefficient of the oxidised state and t_(L) is the reaction half lifebefore the oxidised state is reformed. It will be appreciated thatbecause the size of the micro electrode is small in comparison to thisdiffusion distance, there is normally very little current enhancement inthe presence of the catalytic reaction. In other words, the presence ofthe analyte which promotes the catalytic reaction leads to only a smallcurrent increase above that initially present. The small magnitude ofthis “perturbation current” thus means that accurate analytical resultscannot be obtained.

SUMMARY OF THE INVENTION

Despite these significant drawbacks it has surprisingly been found thatif the determination is made after the reaction has taken place theshape of the potential curve after an initial peak is substantiallyhorizontal with the result that, firstly, a direct measurement can beobtained without any need for subtraction for the background and that,secondly, only one measurement is required. FIG. 1 illustrates a typicalcurve which can be obtained. In other words, the concentration can bedetermined directly from a single value.

According to the present invention there is provided a method fordetermining the concentration of an analyte in a sample which comprisescontacting the sample with a micro electrode which comprises an enzymecapable of reacting with said analyte and a redox mediator which iscapable of being converted by being oxidised or reduced by said enzymeonce the latter has reacted with the analyte, allowing the analyte toreact with the enzyme, then applying a potential across the electrode,and measuring the resulting concentration of the converted mediatorelectrochemically. Typically, the resulting current can be measured andthe concentration determined directly by comparison to a storedreference data set. A catalytic current is generated as the enzymecontinually turns over the substrate and the enzyme is continuallyreused. According to the present invention, the reaction goes tocompletion or reaches a stable equilibrium state which might be lessthan 100% conversion and could be as little as, say, 50% conversionbefore the potential is applied; the drive to complete the reactionarises from the vast excess of the enzyme in this system—many orders ofmagnitude bigger than in the catalytic system.

It will be appreciated that with such a method reaction within themediator occurs throughout the solution, the mediator reacts with theenzyme and when the voltage is applied the resulting oxidised/reducedmediator is reduced/oxidised to its original oxidation state at theelectrode. This is in contrast to a catalytic system where the mediatoris oxidised/reduced and this then reacts with the enzyme—a catalyticamount is obtained with a low level of enzyme throughout the solution isdue to continued turnover. It follows that in the system of the presentinvention the enzyme should be in excess throughout the whole of thesolution which is under test. In contrast in the normal catalytic systemas the reaction is confined to the solution adjacent to the electrodesurface the bulk of the test solution is unperturbed by the enzymaticreaction. It should be noted this requirement does not necessarily meanthat the enzyme has to be homogenous through the mixture. Practically ithas been found that the enzyme excess should desirably be such that thepredicted reaction time is an order of magnitude lower than theacceptable measurement time for the sensor. Hence the required amount ofenzyme dried on a strip (which will be related to the wet-up time) willbe dictated by the required response time, the rate of enzymere-suspension along with the amount of activity retained by the enzyme,the volume in which the test is being completed and the maximumconcentration of analyte to be tested. Clearly these parameters willvary from both test to test and enzyme to enzyme. For example, if theenzyme has an activity of 1000 U/mg, the reaction volume is 10 μL, therequired response time is 10 seconds, 50% of the enzyme activity isrecovered on re-suspension and the maximum analyte concentration is 1mM, then 2 U/ml are required in the deposition solution. It will beappreciated that the reacted mediator is present at a concentrationcorresponding to an electron ratio of 1:1 with the analyte.

Conventional microelectrodes, typically with a working electrode and areference electrode can be used in the method of the present inventionso that a detailed discussion of them is unnecessary. In a preferredembodiment the working electrode is in a wall of the receptable formingthe micro electrode as disclosed in British Application No. 0130684.4,to which reference should be made for further details. Likewise theusual enzymes and mediators can be employed. Typical mediators thusinclude ferricyanide, phenazine alkoxysulphates such phenazineethosulphate and phenazine methosulphate and substituted phenazinealkoxysulphates including 1-methoxy phenazine methosulphate along withphenylene diamine and ruthenium compounds such as ruthenium hexamine.Suitable enzymes which can be used will, of course, depend on theanalyte and on the mediator. By way of example, suitable enzymes whichcan be used with ferricyanide include glucose dehydrogenase (forglucose), cholesterol esterase, horseradish peroxidase, cholesteroldehydrogenase and cholesterol oxidase for cholesterol, lipo proteinlipase, glycerol kinase and glycerol-3-phosphate oxidase fortriglycerides, lactate oxidase and lactate dehydrogenase for lactate aswell as diaphorase. The normal stabilisers for the enzymes such as BSA(bovine serum albumen) as well as non-ionic polyol surfactants such asthose known under the trade mark Triton X and cholic acid and other bileacid salts can be used. Typically the mediator is used in aconcentration from 0.01 to 1 molar, such as 0.05 to 0.25 molar while theconcentration of enzyme is typically 10 to 10⁶ U/ml, for example 100 to10,000 U/ml. The mediator should normally be in excess in relation tothe analyte. Desirably the pH is controlled by the addition of bufferssuch as potassium phosphate so that the pH is maintained at the optimumlevel for the particular analyte under test. Other buffers which can beused include sodium phosphate, Goods buffer,tris(hydroxymethyl)aminomethane (Tris), citrate/phosphate,3-morpholinopropanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid(MES), N-2-hydroxyethyl piperazine (HEPES), tricine, bicine,piperazine-N,N′-bis(2-ethane sulfonic acid) (PIPES),N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES),3-cyclohexylamino)-1-propanesulfonic acid (CAPS) and[(2-hydroxy-1,1-bis[hydroxymethyl]ethyl)amino]-1-propanesulfonic acid(TAPS) as well as other biological buffers.

The micro electrodes typically comprise a working electrode and acounter electrode as well as a reference electrode. In some cases thecounter electrode and the reference electrode are combined. The workingelectrode is typically made of palladium, platinum, gold or carbon. Thecounter electrode is typically carbon, Ag/AgCl, Ag/Ag₂SO₄, palladium,gold, platinum, Cu/CuSO₄, Hg/HgO, Hg/HgCl₂, Hg/HgSO₄ or Zu/ZuSO₄.

When enzymes are deposited on a surface such as an electrode they have atendency to both denature and to form relatively insoluble “masses”.Typically, the enzyme solution is dried on the sensor and, accordingly,these issues need to be addressed to ensure the enzyme retains itsactivity and that it re-suspends within a reasonable timescale. It hasbeen found that this reaction can be assisted by depositing the enzymesolution on the electrode in the presence of salt such as a chloride orsulphate wherein the cation is typically potassium, ammonium, ormagnesium such as potassium and magnesium chloride and ammoniumsulphate, or detergent such as those known under the trade mark Triton Xe.g. Triton X-100 or sodium deoxycholate and similar bile acid salts,SDS, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), 3[(3-cholamidopropyl)dimethylammonio]propanesulfonic acids(CHAPS), octylglucopyranoside, octylthiogluconpyranoside and variousother polyols, polyvinylalcohol (PVA), polyethylene glycol (PEG),carboxymethyl cellulose, dextran sulphate, hydroxypropylmethylcellulose, starch, n-dodecyl maltoside, ethyl cellulose andpolymethacrylic acid or microcrystals such as sepharose, Sephadex G25.Alternatively, one or more of these components can be applied to theelectrode surface first and then dried before the enzyme solution isapplied.

In a typical measurement, after the sample has been applied to the microelectrode a time (SD) of, for example, 0.1 or 1 to 180, preferably 0.1,0.5 or 1 or even 10 to 60, more preferably 0.1 to 60, especially 0.5 to20, in particular about 20 seconds, is set during which all theelectrodes are held at earth potential i.e. no current flows. This is toallow time for the enzyme to re-suspend, to substantially react fully(or reach a stable equilibrium state) with the analyte and for theelectrodes and enzyme to become fully wetted. A potential, typicallyfrom −2 to +2V, e.g. from −1 to +1V, for example about 0.5V, dependingon the enzyme system employed is then applied to the electrode and thisdetermines the amount of mediator stochiometrically turned over by theenzyme. A time delay (D1), typically of 10 to 500 milliseconds andgenerally no more than 1 or 5 seconds, is made prior to making ameasurement. Typically the measurement (C1) is the current response.After this time delay the current is measured. It will be appreciatedthat there is no need to know the measurement when the potential isfirst applied.

The device can be calibrated beforehand with given concentrations ofanalyte so that a direct reading can be obtained. It has been found tobe that the current obtained is proportional to the concentration of theanalyte.

Although a single reading is all that is necessary, in general it isdesirable that more than one reading, for example 10 to 100 readings ata sample state of, say, 25 to 1000 Hz, is obtained in order to eliminateerror and obtain an average value. It is important to stress, though,that these multiple readings are taken purely for averaging purposes incontrast to the situation when using a macro electrode where multiplereadings are essential in order to obtain a concentration value by meansof integration or a specific algorithm.

It has been found, though, that there is a difficulty in pre-calibratinga micro electrode even, though, ostensibly it is made to be identical toa previous micro electrode. In other words the pre-calibration of theprevious micro electrode cannot be used accurately for the subsequentmicro electrode. Because of the small sizes involved it is verydifficult to ensure that the area of the electrode, which does, ofcourse, critically determine the current which is obtained, is preciselythe same from one micro electrode to the next. Use of the areacompensation technique will eliminate errors induced due to differentelectrode sizes in the short timescale regime. Differences in electrodearea will also result in the smaller areas reaching a quasi-steady statefaster than the relatively larger areas. This variation will be apparentin the relative increase in magnitude of the electrochemical response.In the case of the current response; this is exacerbated if the systemis operated at short timescale by the current due to planar diffusionadding to the quasi-steady state current but will frequently be apparentalso at longer timescales when the electrodes have reached a steadyrate.

The use of an independent calibration sample is useful in this regardand also to eliminate any differences arising out of changes in thewetting-up volume. Under some circumstances it may be best to add aseparate redox probe as a calibrator; judicious choice of this probe isessential to ensure that (a) the initial redox state of the probe issuch that it is thermodynamically unfavourable for it to react with anyredox state of the mediator (b) any reaction between any subsequentredox state of the redox probe and the mediator is too slow to observeon the timescale of our measurement.

According to a particular aspect of the present invention, a means hasbeen found of eliminating any error due to differences in the area ofthe electrode. This involves a particular method of working. For anamperometric measurement, effectively after the measurement has beenobtained (i1) as described above, for example after a time delay of,say, 10 to 500 milliseconds, as before, the potential is reversed sothat the mediator which has been oxidised by the reaction is thenreduced and another value (C2) of the current obtained (i2), typicallyafter a similar time delay (D3). It will be appreciated that in eachcase the current obtained is proportional to the area of the electrodesurface so that by obtaining a ratio of the two currents (i1/i2) or apercentage of the first current in relation to the sum of the twocurrents (i1/i1+i2) a value can be obtained which is independent of thesurface area of the electrode FIG. 3 illustrates, diagrammatically, theprocedure. This ratio or percentage can therefore be read off directlyfrom a calibration curve in exactly the same way as if a singlemeasurement is obtained. For a voltametric measurement using a givenredox couple, a second non-reactive redox couple can be used; it isimportant that the relative concentration of the two redox couplesremain constant across all the electrodes under test.

This compensation method of working is, therefore, of particular valuein eliminating differences in the surface area of micro electrodes beingproduced, typically as biosensors. Since the observed current is alinear combination of planar and ‘radial’ diffusion the use of the ratiotechnique can eliminate changes in relative percentages of these twocomponents resulting from the different electrode sizes.

Accordingly, the present invention also provides a method fordetermining the concentration of an analyte in a sample which comprisescontacting the sample with a micro electrode which contains an enzymecapable of reacting with said analyte and a redox mediator which iscapable of being converted by being oxidised or reduced by said enzymeonce the latter has reacted with the analyte, allowing the analyte toreact with the enzyme, then applying a potential across the electrode,measuring the resulting current, reversing the potential and measuringthe current again, expressing the two currents as a ratio or apercentage and determining the concentration of the analyte directlytherefrom.

In order to simplify the operation, the present invention also providesa micro electrode comprising a working electrode and a counterelectrode, means for applying a positive or negative potential acrossthe electrode at a given time after the sample has been applied to it,means for determining the resulting current at a set time of no greaterthan 1 second, e.g. from to 500 milliseconds, thereafter, means forreversing the potential across the electrode and determining theresulting current at said set time after the reversal. It will beappreciated that the various means can be provided by a singleprocessor/microchip.

The following Examples further illustrate the present invention. Thefollowing sensors have been used:

Cholesterol Oxidase Sensor:

-   Horseradish peroxidase @ 400 U/mL-   Cholesterol oxidase @ 700 U/ml-   Cholesterol esterase @ 700 U/ml-   0.08 M potassium ferricyanide-   0.1 M potassium chloride-   0.1 M potassium phosphate, pH 7.4-   100 g/dm⁻³ Triton-X 100-   0.2 gm mL Sephadex G25    Triglyceride Sensor:-   Glycerol phosphate oxidase @ 4500 U/mL-   Glycerol kinase @ 4500 U/mL-   Lipase @100000 U/mL-   0-2 M potassium chloride-   0.2 potassium ferricyanide-   0.025 M adenosine triphosphate-   0.002 M ammonium sulphate-   0.002 M magnesium chloride-   0.1 g/mL Sephadex G25

If plotted against glycerol concentration, the first oxidative currentdepends on enzyme turnover but also on electrode surface area. Becausedisposable microelectrodes tend to have variable surface areas, therelation between oxidation current and glycerol concentration iscompromised.

On the other hand, by dividing the oxidation current from the firstpotential step by the reduction current from the second potential step,a unitless ratio is obtained which eliminates the electrode area factor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the current response during the double potential stepexperiments. The value of the current is seen to become constant withinabout 400 msec from the potential step. The current values for theoxidation step and the reduction currents are read from the plot 400msec after the first and the second potential step respectively.

FIG. 2 shows oxidation currents i.e. from the first potential step only,plotted against glycerol concentrations. These currents are subject toelectrode area variability.

FIG. 3 is a plot of ratios obtained from the oxidation currentsdisplayed in FIG. 2, divided by their respective reduction currentsobtained from the second potential step (as shown in FIG. 1). Theseratios are the result of an area compensation and prove to be linearwith glycerol concentration.

FIG. 4 shows oxidation currents plotted against glucose concentrationsusing the specific system described in Example 1.

FIG. 5 shows oxidation currents plotted against LDL cholesterolconcentrations using the specific system described in Example 4.

FIG. 6 shows oxidation currents plotted against NADH concentrationsusing the specific system described in Example 5.

FIG. 7 shows oxidation currents plotted against NADH concentraions usingthe specific system described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Measurements were made on an electrodes of the type disclosed in Britishapplication no. 0130684.4. The working electrodes were Coates carbonprinted onto a 250 μm melonex upper substrate. This was adhered to a 125μm melonex lower substrate in order to form the well. Ag/AgCl wereprinted onto the upper substrate. The components of the biosensorcoating solution (concentrations) were hexaammineruthenium (III)chloride (4.8 mM), NAD+ (0.8 mM), PdR (1.6 μM), polymeric detergent (2.5mM) and glucose dehydrogenase (1 U/biosensor).

The supporting electrolyte was 0.1M, pH 7.4 phosphate. A potential of+0.2V versus Ag/AgCl was applied and measurements were taken 0.5 s afterthe application of the potential. The results obtained are shown in FIG.4.

EXAMPLE 2

Electrodes were constructed as in Example 1 from a 250 μm PET layer onwhich a 7 μm Coates carbon ink layer has been screen-printed followed bya 30 μm Dupont 5036 dielectric layer. This layer has been punched toproduce a 1 mm diameter hole and has then been adhered to a 125 μm PETbase layer using pressure sensitive lamination, with a common Ag/AgCl(using Ercon E0430-128) counter reference on the top of the strip.

Amperometric current was measured 1 second after the application 0.15 Vfollowed by the application of −0.45 V vs. Ag/AgCl on the addition ofvarying amounts of LDL cholesterol in 0.1 mol dm⁻³ Tris buffer at pH 7.4containing 0.1 mol dm⁻³ KCl to electrodes on which 0.3 μL of a solutioncontaining NAD (0.022 g/ml, ruthenium hexaamine @ 0.021 g/ml,cholesterol esterase @ 1.25 kU/ml, cholesterol dehydrogenase @ 4.2kU/ml, putidaredoxin reductase @ 650 kU/ml, 0.1 M KCL, 0.1 M Tris-HCl @pH 9 octylglucopyranoside @ 100 g/dm⁻³ has been dried.

The results obtained are given in the following table:

Without area With area compensation compensation LDL/mM Current/nA % CVRatio % CV 1 285 15 5.36 0.6 3 354 13 8.33 2.8 5 691 10 15.3 6.3

It can be seen that the use of area compensation decreases the magnitudeof the error and is therefore extremely useful in the production ofaccurate sensors.

EXAMPLE 3

Electrodes were constructed as in Example 1 from a 250 μm PET layer onwhich a 20 μm Coates 268203 carbon ink layer had been screen-printed,followed by a 30 μm Ronseal dielectric layer. This layer has beenpunched to produce a 1 mm diameter hole and has then been adhered to thePET base layer using 7841 sheet adhesive, with a common Ag/AgCl (ErconE0430-128) counter reference on the top of the strip.

Amperometric current was measured 1 second after the application of0.20V vs. Ag/AgCl on the addition of 2, 5, 7.5, 10, 12.5 and 15 mmoldm⁻³ glycerol in 0.1 mmol dm⁻³ Tris buffer at pH 9, containing 0.1 moldm⁻³ KCl and 1% OGP, to electrodes on which 0.3 μL of a solutioncontaining 0.1 mol dm⁻³ ruthenium hexamine, 0.15 mol dm⁻³ ammoniumsulfate, 0.04 mol dm⁻³ NAD, 150 U/mL glycerol dehydrogenase and 6.7kU/mL diaphorase have been dried.

The results obtained are shown in the following table:

Oxidation Reduction [Glycerol]/mM Current, nA current, nA Ratio 2.0 95−3815 2.50 5.0 167 −3772 4.42 5.0 185 −3776 4.91 7.5 177 −2288 7.75 7.5192 −2827 6.80 7.5 163 −2426 6.70 7.5 224 −3217 6.97 10.0 343 −3769 9.1012.5 226 −2098 10.77 12.5 283 −2400 11.80 12.5 233 −2358 9.90 12.5 277−2888 9.59 15.0 433 −3582 12.09

EXAMPLE 4

Electrodes were constructed as in Example 1 from a 250 μm PET layer onwhich a 7 μm Coates carbon ink 268203 layer has been screen-printedfollowed by a 30 μm Dupont 5036 dielectric layer. This layer has beenpunched to produce a 1 nm diameter hole and has then been adhered to a125 μm PET base layer using pressure sensitive lamination, with a commonAg/AgCl (using Ercon E0430-128) counter reference on the top of thestrip.

Amperometric current was measured 1 second after the application 0.15 Vfollowed by the application of −0.45 V vs. Ag/AgCl on the addition of 1,3, 5 mmol dm⁻³ LDL cholesterol in 0.1 mol dm⁻³ Tris buffer at pH 7.4containing 0.1 mol dm⁻³ KCl to electrodes on which 0.3 μL of a solutioncontaining NAD @ 0.022 g/ml, ruthenium hexaamine @ 0.021 g/ml,cholesterol esterase @ 1.25 kU/ml, cholesterol dehydrogenase @ 4.2kU/ml, putidaredoxin reductase (650 kU/ml, 0.1 M KCL, 0.1 M Tris-HCl (pH9 octylglucopyranoside @ 100 g/dm⁻³ has been dried.

The results obtained are shown in FIG. 5.

EXAMPLE 5

Electrodes were constructed as in Example 1 from a 250 μm PET layer onwhich a 15 μm Coates carbon ink 26-8203 layer has been screen-printedfollowed by a 30 μm Ronseal layer. This layer has been punched toproduce a 1 mm diameter hole and has then been adhered to a 125 μm PETbase layer using ARcare 7841 sheet adhesive, with a common Ag/AgClcounter reference on the top of the strip.

Cyclic voltammetric current was measured at 0.15 V vs. Ag/AgClimmediately after addition of 2, 4, 6, 8 and 10 mmol dm⁻³ NADH in 0.1mol dm⁻³ Tris buffer at pH 9 containing 0.1 mol dm⁻³ KCl to electrodeson which 0.2 μL of a solution containing 0.2 mol dm⁻³ rutheniumhexaamine and 650 KU/mL putidaredoxin reductase has been dried.

The results obtained are shown in FIG. 6.

EXAMPLE 6

Electrodes were constructed as in Example 1 from a 250 μm PET layer onwhich a 15 μm Coates carbon ink 26-8203 layer has been screen-printedfollowed by a 30 μm Ronseal layer. This layer has been punched toproduce a 1 mm diameter hole and has then been adhered to a 125 μm PETbase layer using ARcare 7841 sheet adhesive, with a common Ag/AgClcounter reference on the top of the strip.

Amperometric current was measured 1 second after the application 0.15 Vvs. Ag/AgCl on the addition of 2, 4, 6, 8 and 10 mmol dm⁻³ NADH in 0.1mol dm⁻³ Tris buffer at pH 9 containing 0.1 mol dm⁻³ KCl to electrodeson which 0.2 μL of a solution containing 0.2 mol dm⁻³ rutheniumhexaamine and 650 KU/mL putidaredoxin reductase has been dried.

The results obtained are shown in FIG. 7.

1. A method for determining the concentration of an analyte in a samplewhich comprises contacting the sample with a micro electrode whichcomprises an enzyme capable of reacting with said analyte and a redoxmediator which is capable of being converted by being oxidised orreduced by said enzyme once the latter has reacted with the analyte,allowing the analyte to react with the enzyme, said reaction going tocompletion or substantially to completion before a potential is applied,then applying a potential across the electrode and measuring theresulting concentration of the converted mediator electrochemically. 2.A method according to claim 1 wherein the resulting concentration ismeasured from the resulting current.
 3. A method according to claim 1wherein the micro electrode comprises a working electrode, a counterelectrode and a reference electrode.
 4. A method according to claim 3wherein the counter electrode and reference electrode are combined.
 5. Amethod according to claim 3 wherein the working electrode is made ofpalladium, platinum, gold or carbon and the counter electrode is made ofcarbon, Ag/AgCl, Ag/Ag₂SO₄, palladium, gold, platinum, Cu/CuSo₄, Hg/HgO,Hg/HgCl₂, Hg/HgSO₄, or Zu/ZuSO₄.
 6. A method according to claim 1wherein the mediator is ferricyanide, phenazine ethosulphate, phenazinemethosulphate, 1-methoxy phenazine methosulphate, phenylene diamine orruthenium hexamine.
 7. A method according to claim 1 wherein the enzymeis glucose dehydrogenase, cholesterol esterase, horseradish peroxidase,cholesterol dehydrogenase, cholesterol oxidase, lipo protein lipase,glycerol kinase, glycerol dehydrogenase, glycerol-3-phosphate oxidase,lactate oxidase or lactate dehydrogenase.
 8. A method according to claim1 which comprises allowing a time of 0.5 to 180 seconds to elapse afterthe sample has been applied to the micro electrode before applying apotential across the electrode and making an electrochemical measurementno more than 5 seconds thereafter.
 9. A method according to claim 8wherein the potential applied is from −2 to +2 volt.
 10. A methodaccording to claim 8 wherein the measurement is taken 10 to 500milliseconds after the potential has been applied.
 11. A methodaccording to claim 8 wherein 10 to 100 measurements are taken.
 12. Amethod according to claim 1 wherein the micro electrode has previouslybeen calibrated to provide a direct reading.
 13. A method according toclaim 1 wherein the mediator is present in excess in relation to theanalyte.
 14. A method of determining the concentration of an analyte ina sample which comprises contacting the sample with a micro electrodewhich contains an enzyme capable of reacting with said analyte and aredox mediator which is capable of being converted by being oxidised orreduced by said enzyme once the latter has reacted with the analyte,allowing the analyte to react with the enzyme, then applying a potentialacross the electrode, measuring the resulting current, reversing thepotential and measuring the current again, expressing the two currentsas a ratio or a percentage and determining the concentration of theanalyte directly therefrom.
 15. A method according to claim 14 whereinthe micro electrode comprises a working electrode, a counter electrodeand a reference electrode.
 16. A method according to claim 15 whereinthe counter electrode and reference electrode are combined.
 17. A methodaccording to claim 15 wherein the working electrode is made ofpalladium, platinum, gold or carbon and the counter electrode is made ofcarbon, Ag/AgCl, Ag/Ag₂SO₄, palladium, gold, platinum, Cu/CuSo₄, Hg/HgO,Hg/HgCl₂, Hg/HgSO₄, or Zu/ZuSO₄.
 18. A method according to claim 14wherein the mediator is ferricyanide, phenazine ethosulphate, phenazinemethosulphate, 1-methoxy phenazine methosulphate, phenylene diamine orruthenium hexamine.
 19. A method according to claim 14 wherein theenzyme is glucose dehydrogenase, cholesterol esterase, horseradishperoxidase, cholesterol dehydrogenase, cholesterol oxidase, lipo proteinlipase, glycerol kinase, glycerol dehydrogenase, glycerol-3-phosphateoxidase, lactate oxidase or lactate dehydrogenase.
 20. A methodaccording to claim 14 which comprises allowing a time of 0.5 to 60seconds to elapse after the sample has been applied to the microelectrode before applying a potential across the electrode and making anelectrochemical measurement no more than 5 seconds thereafter.
 21. Amethod according to claim 20 wherein the potential applied is from −2 to+2 volt.
 22. A method according to claim 20 wherein the measurement istaken 10 to 500 milliseconds after the potential has been applied.
 23. Amethod according to claim 20 wherein 10 to 100 measurements are taken.24. A method according to claim 14 wherein the micro electrode haspreviously been calibrated to provide a direct reading.
 25. A methodaccording to claim 14 wherein the mediator is present in excess inrelation to the analyte.